Microarrays for Methylation

ByRobert M. Frederickson

Dec. 2006 / Jan. 2007 | Epigenetics is a regulatory process that is not necessarily dictated by the order of the DNA sequence, but instead by how the sequence is interpreted or modified in the cell. For example, methylation of cytosine residues in a cytosine-guanine (CG) pair is one important epigenetic mechanism that has been linked to gene silencing.

It turns out that gene methylation is a dynamic process that can be affected by a number of environmental factors, resulting in large variations in methylation patterns in our cells. Recent data further suggests that altered gene methylation patterns contribute to tumor development. Thus, there is a strong need and demand for reliable methods to scan tumor samples for changes in patterns of gene methylation.

Current methods involve chemical conversion of unmethylated cytosines using bisulfite or the use of restriction enzymes that cut DNA in a methylation-dependent manner. Both have drawbacks: the bisulfite method is able to determine methylation of specific CG pairs, but less able to do comprehensive analysis; enzymatic approaches are best at quantifying overall densities of methylation.

Discovery ToolOrion Genomics has developed two methods for enzymatic DNA methylation screening. MethylScope is a DNA array-based technology that can be used to screen methylation patterns of the entire genome for potential new epigenetic markers. Published in Nature in 2004, MethylScope technology is subject to numerous patent applications by Cold Spring Harbor Laboratory and Orion.

A single MethylScope microarray can quantitatively detect the methylation status of every human gene. DNA from a tissue or cell of interest is labeled with different colors to distinguish methylated from unmethylated fragments. The fragments are hybridized to the MethylScope array, scanned, and a "methylation score" is given to every gene on the array. Comparing methylation profiles between samples permits the identification biomarkers associated with diseases. So far, MethylScope technology is the only platform able to detect DNA methylation for all human genes on a single array.

MethylScreen, in contrast, is a PCR-based assay that can be used for clinical diagnostics. This single-step technology is sensitive and amenable to high-throughput operation. The assay can detect trace amounts of specific methylated biomarkers in a background of normal DNA, such as would be required in a serum test for cancer. The assay also quantifies the relative methylation density within each detected biomarker.

MethylScreen is based on DNA digestion with methylation-sensitive enzymes, and real-time quantitative PCR. For each biomarker locus, the assay measures both unmethylated and methylated copies with redundant checks. This information is used to identify several different classes of methylated biomarkers, which can then be correlated to a particular disease, the stage of disease, and/or the response of a patient to therapeutics. Automated instruments suitable for MethylScreen assays already exist in most reference laboratories.

In October, Orion announced a collaboration with Mayo Clinic to study the clinical utility of Orion's breast cancer screening tests, which are based on epigenetic biomarkers identified using Orion's DNA methylation technologies. The Mayo Clinic and Orion will validate the tumor specificity of Orion's breast cancer biomarkers by analyzing epigenetic biomarkers in more than a dozen additional cancer types. Orion has so far discovered and validated over 50 novel breast cancer biomarkers.

In Good CompanyAnother company working on methylation detection is Panomics, whose technology permits high throughput analysis of promoter methylation, simultaneously profiling the methylation status of 82 promoter regions. The company's TransSignal Array assay involves three steps. First, genomic DNA is digested with a restriction enzyme to isolate CpG islands. Next, the digests are purified, adapted with linkers and incubated with the methylation binding protein (MBP). This forms a protein/DNA complex. Finally, the complexes are separated and methylated DNA is isolated. Methylated DNA is labeled with biotin-dCTP via PCR and these probes are hybridized to a methylation array.

Illumina is also developing a DNA methylation assay it says can analyze up to 1,536 CpG sites per sample and 96 samples at a time. The process is based on Illumina's GoldenGate BeadArray platform and its Standard Methylation Cancer Panel will profile CpG sites from about 800 cancer genes. The GoldenGate Assay is based on high-throughput genotyping of bisulfite-converted genomic DNA.